ARPE-19 as a platform cell line for encapsulated cell-based delivery

ABSTRACT

ARPE-19 cells were evaluated as a platform cell line for encapsulated and un-encapsulated cell-based delivery technology. ARPE-19 cells were found to be hardy (the cell line is viable under stringent conditions, such as in central nervous system or intra-ocular environment); can be genetically modified to secrete the protein of choice; has a long life span; is of human origin; has good in vivo device viability; delivers efficacious quantity of growth factor; triggers no or low level host immune reaction, and is non-tumorigenic.

CLAIM OF PRIORITY

This application claims priority to U.S. provisional patent applicationNo. 60/127,926, filed Apr. 6, 1999.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to cellular therapy and encapsulateddevices.

BACKGROUND OF THE INVENTION

Growth factors have tremendous therapeutic potential forneurodegenerative disorders. However, growth factors have yet to besuccessfully developed into clinical treatments due to the fact thatlarge proteins, like growth factors, do not cross the blood-brainbarrier. The transplantation of cells genetically engineered to producegrowth factors offers a partial solution to the problem of growth factordelivery, because grafts of growth factor-producing cells can bypass theblood-brain barrier and deliver the therapeutic factors directly to thetarget site. Unfortunately, these transplants are subject to host immunerejection and require immunosuppression. Also, grafts of somegenetically engineered cell lines can form lethal tumors.

Implanting cells that have been macroencapsulated in semi-permeablepolymer membranes provides a better solution to these problems.Mammalian cells that have been genetically engineered to produce growthfactors can be encapsulated in semipermeable polymer membranes. Thesemipermeable membranes protect the encapsulated cells from acute hostimmune rejection, but allow the delivery of the therapeutic agents intothe host tissue. These small bioartificial devices (cellsmacroencapsulated in semipermeable membranes) can be implanted directlyinto the target site for site-specific, continuous, long-term, low-leveldelivery of the desired factors. Encapsulating cells in semi-permeablemembranes also reduces the risk of tumor development. Furthermore,polymer-encapsulated cell transplants have lower incidences ofinfection, because the transplants require only a single penetrationinto the target site for continuous growth factor delivery.

Regarding the delivery of desired growth factors, pre-clinical studieshave shown that polymer-encapsulated cells can deliver ciliaryneurotrophic factor (CNTF) continuously with therapeutic efficacy inrodent models (Emerich et al., 16 J. Neurosci. 5168-81 (1996)). Clinicaltrials support the safety of chronic CNTF delivery into the humancentral nervous system (CNS) with polymer-encapsulated cells (Aebischeret al., 7 Hum. Gene Ther. 851-60 (1996), Aebischer et al., 2 NatureMedicine 696-9 (1996)). However, a major challenge in translating suchsuccesses from rodent models to humans is ensuring long-term cellviability in encapsulated devices in vivo.

SUMMARY OF THE INVENTION

The ARPE-19 cell line is a superior platform cell line for encapsulatedcell based delivery technology and is also useful for unencapsulatedcell based delivery technology. The ARPE-19 cell line is hardy (i.e.,the cell line is viable under stringent conditions, such as implantationin the central nervous system or the intra-ocular environment). ARPE-19cells can be genetically modified to secrete a substance of therapeuticinterest. ARPE-19 cells have a relatively long life span. ARPE-19 cellsare of human origin. Furthermore, encapsulated ARPE-19 cells have goodin vivo device viability. ARPE-19 cells can deliver an efficaciousquantity of growth factor. ARPE-19 cells elicit a negligible host immunereaction. Moreover, ARPE-19 cells are non-tumorigenic.

The therapeutic usefulness of polymer-encapsulated ARPE-19 cell-baseddelivery of ciliary neurotrophic factor (CNTF) for treatment ofdegenerative diseases was shown in both a rodent and canine model ofretinitis pigmentosa. ARPE-19 cells were genetically modified to secreteCNTF. Encapsulated genetically modified ARPE-19 cells delivered aconsistent amount of CNTF, for example, over a 7-week implantationinterval. Cell viability within the encapsulated devices was excellent.The presence of the encapsulated cell device in the eye caused nosignificant adverse effects on the retina. These results provide a proofof principle for the therapeutic potential of encapsulated ARPE-19cell-based delivery of desired neurotrophic factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing protection of photoreceptors intransgenic rats carrying the rhodopsin mutation S334ter by ARPE-19/CNTFcells. (A) S334ter untreated rat; (B) ARPE-19 parental cell treatedcontrol rat; and (C) ARPE-19/CNTF cell treated rat.

FIG. 2 is a bar graph showing anti-ARPE-19 specific IgG titer in dog vs.human serum. ARPE-19, Hs27, and SIRC cells were incubated with eitherdog or human serum dilutions. The antibody titer was measured by flowcytometric crossmatch (FCXM).

FIG. 3 is a bar graph showing cytotoxic ARPE-19 specific antibody titerin dog vs. human serum. ARPE-19, Hs27, and SIRC cells were incubatedwith either dog or human serum dilutions. The cytotoxic antibody titerwas measured by complement dependent cytotoxicity crossmatch (CDC).

DETAILED DESCRIPTION OF THE INVENTION

A. ARPE-19 Cell Line as a Platform Cell Line for an Encapsulated CellBased Delivery System

To be a platform cell line for an encapsulated cell based deliverysystem, the cell line should have as many of the followingcharacteristics as possible: (1) The cells should be hardy understringent conditions (the encapsulated cells should be functional in theavascular tissue cavities such as in the central nervous system or theeye, especially in the intra-ocular environment). (2) The cells shouldbe able to be genetically modified (the desired therapeutic factorsneeded to be engineered into the cells). (3) The cells should have arelatively long life span (the cells should produce sufficient progeniesto be banked, characterized, engineered, safety tested and clinical lotmanufactured). (4) The cells should preferably be of human origin (whichincreases compatibility between the encapsulated cells and the host).(5) The cells should exhibit greater than 80% viability for a period ofmore than one month in vivo in device (which ensures long-termdelivery). (6) The encapsulated cells should deliver an efficaciousquantity of a useful biological product (which ensures effectiveness ofthe treatment). (7) The cells should have a low level of host immunereaction (which ensures the longevity of the graft). (8) The cellsshould be nontumorigenic (to provide added safety to the host, in caseof device leakage).

We screened and characterized several cell lines, identified optimalencapsulated cell device configurations, and evaluated cell viabilitieswithin devices in different animal models. We found that the ARPE-19cell line (Dunn et al., 62 Exp. Eye Res. 155-69 (1996), Dunn et al., 39Invest. Ophthalmol. Vis. Sci. 2744-9 (1998), Finnemann et al., 94 Proc.Natl. Acad. Sci. USA 12932-7 (1997), Handa et al., 66 Exp. Eye. 411-9(1998), Holtkamp et al., 112 Clin. Exp. Immunol. 34-43 (1998), Maidji etal., 70 J. Virol. 8402-10 (1996)) had all of the characteristics of asuccessful platform cell for an encapsulated cell-based delivery system.The ARPE-19 cell line was superior to the other cell lines that wetested.

The ARPE-19 cell line is available from the American Type CultureCollection (ATCC Number CRL-2302). ARPE-19 cells are normal retinalpigmented epithelial (RPE) cells and express the retinal pigmentaryepithelial cell-specific markers CRALBP and RPE-65. ARPE-19 cells formstable monolayers, which exhibit morphological and functional polarity.

ARPE-19 cells are cultured in Complete Growth Medium, theserum-containing medium recommended by the cell depositor. CompleteGrowth Medium is either a 1:1 mixture of Dulbecco's modified Eagle'smedium and Ham's F12 medium with 3 mM L-glutamine, 90%; fetal bovineserum, 10% or a 1:1 mixture of Dulbecco's modified Eagle's medium andHam's F12 medium with HEPES buffer containing 10% fetal bovine serum, 56mM final concentration sodium bicarbonate and 2 mM L-glutamine andincubated at 37° C. in 5% CO₂. The cells were plated. The cells weretypically grown in Falcon tissue culture treated 6 or 12-well plates orT25 or T75 flasks.

For subculturing, spent medium is removed, and the ARPE-19 cells arerinsed with 0.05% trypsin, 0.02% EDTA solution, and the trypsin isremoved. One to two ml of additional trypsin solution is added. Theculture is incubated at room temperature (or at 37° C.) until theARPE-19 cells detach. A subcultivation ratio of 1:3 to 1:5 isrecommended.

1. The ARPE-19 cell line is hardy.

To evaluate the hardiness of cell lines, a three-step screen wasestablished. (a) Cell viability screen (The cells were evaluated understressed conditions using artificial aqueous humor (aAH) medium orartificial cerebral spinal fluid (aCSF) medium). (b) In vitro ECM screen(The cells were evaluated in an in vitro extra-cellular matrix (ECM)screen). (c) In vivo device viability screen (The encapsulated cellswere evaluated in an in vivo membrane screen).

(a) Cell viability in vitro screen. Effect of aAH and aCSF on test cells(including ARPE-19 cells) was examined. Artificial aqueous humor (aAH)and artificial cerebral spinal fluid (aCSF) were formulated according tothe protocols from Geigy Scientific Tables. The detailed formulationsare listed in TABLE 1:

TABLE 1 FORMULATIONS FOR AAH & ACSF Artificial Artificial AqueousCerebral Humor Spinal (aAH) Fluid (aCSF) (mg/L) (mg/L) Amino Acids (μM)(μM) L-Alanine 294 26.19 28 2.4945 L-α-Amino 31.4 3.24 butyric acidL-Argin- 133 28.02 19 4.0033 ine.HCI L-Aspara- 49.2 6.49 6.3 0.9456 gineL-Aspartic 1 0.13 0.45 0.0599 acid L-Citrulline 9.8 1.72 L-Cys- 0.10.0313 tine.2HCl Ethanol- 16.7 1.02 amine L-Glutamic 12 1.77 11.4 1.6769acid L-Glutamine 717 104.75 515.7 75.3438  Glycine 16.7 1.25 6.2 0.4654L-Histadine. 77.9 16.33 12.6 2.6410 HCI.H₂O L-Isoleucine 79.8 10.47 4.60.6035 L-Leucine 192 25.19 12 1.5744 L-Lysine. 155 28.30 24.9 4.5467 HCIL-Methio- 44.4 6.62 2.7 0.4028 nine L-Orni- 25.3 4.27 4.5 0.7587thine.HCI L-Phenyl- 119 19.66 7.8 0.2886 alanine O-Phosphor- 5.2 0.7337ylethanol- amine L-Proline 16 1.84 L-Serine 179 18.81 25.4 2.6695Taurine 39.1 4.89 L-Threonine 152 18.10 28.8 3.4301 L-Trypto- 31.9 6.511.2 0.2450 phan L-Tyrosine 123 22.26 7.2 0.6214 L-Valine 388 45.43 17.32.0258 Inorganic Salts (mM) (mM) Sodium 21.6 22.6 1898.626 BicarbonateSodium 120 Chloride Potassium 3.2 4 298.2000 Chloride Sodium 111 0.2534.5000 Phosphate Cupric 0.002 0.00025 0.0624 Sulfate Copper 0.0028 ZincSulfate 1.0 0.00064 0.1826 NaF 0.005 0.2100 MnCI2 0.1 0.000021 0.00415MgCI2 0.5 101.6500 KI 0.000016 0.002656 KBr 0.029 3.4510 Short-ChainFatty Acids (mM) Urea 4.51 270.87 DL-Lactic 4.69 422.48 1.47 mM 132.4176acid Citric acid 0.133 25.55 0.176 mM 33.8096 Pyruvic acid 0.14 mM11.9762 Acetic acid 116 μM 7.0830 Propionic 2.8 μM 0.2074 acidIsobutyric 1.8 μM 0.1586 acid n-Butyric 1.4 μM 0.1234 acid Isovaleric1.35 μM 0.1378 acid α-Oxoglu- 8.9 μM 1.3000 taric acid Oxalacetic 7.2 μM0.9511 acid Succinic acid 28.5 μM 3.3659 Ketone 117 μM 6.7954 Bodies (asacetone) Acetoacetic 26.2 μM 2.6700 acid DL-B-Hy- 46.4 μM 5.5810droxybutyric acid Vitamins Nicotin- 40 nmol/ 2.44 amide eye Trigonelline70 nmol/ 6.08 eye B12 22.1 pM 0.0000299 13.58 pM 1.8406E-05 L-Ascorbic810 pM 234.53 60 μM 10.5660 acid Nicotinic 2.4 μM 0.2954 acidD-Pantothen- 2.37 μM 0.5648 ic acid Thiamine 15 nM 0.0050595 (B1) B6 2.2nM 0.00045232 (pyridoxine) Folic acid 62.55 nM 0.02760957 Biopterin 4.3nM 0.00101996 Lipids Triglycerides 4.17 mg/L 4.1700 Cholesterol 4.63mg/L 4.6300 Phospho- 5.49 mg/L 5.4900 lopids Fatty acids 7.36 mg/L7.3600 Prosta- 1.31 nM 0.0004644 glandin PGE1 Prosta- 1.98 nM 0.00094169glandin PGF2a Carbo- hydrates D(+)- 0.756 g/L 756.84 Glucose TotalProtein FBS 10.6 ml/L 450.00 8.4 ml/L Transferrin 59 mg/L 59.00

The cells were plated initially in serum containing medium to allow thecells to attach. The cells were plated at 60,000/cm², and incubated in a37° C. incubator with 5% CO₂. After the cells had attached (18-24hours), the growth medium was removed and the cells were washed 2× withHank's balanced salt solution (HBSS) to remove serum. The medium wasreplaced with either aAH or aCSF (pre-warmed to 37° C.). Spent aAH oraCSF was replaced with fresh aAH or aCSF every other day (Monday,Wednesday, Friday schedule). The test cells were evaluated daily formorphology, viability and % confluency (compared to control) bymicroscopic examination. All cells were evaluated for a one-weekduration. The observations were documented with photographs.

A control of each cell line in complete growth medium was carriedthroughout the screen for comparison with the experimental group. At theend of the test period, the results were recorded in number of cells and% confluency with a note about morphology. Also, viability staining suchas fluorescein diacetate/propidium iodide (FDA-PI) was used to accessviability of the culture. Cells that passed initial cell viability invitro screen proceeded to the in vitro ECM screen.

(b) In vitro extra-cellular matrix (ECM) screen. Cell survival usingvarious extra cellular matrices (ECM) was evaluated under aCSF andhypoxic conditions and the ECM that provided the best survival wasselected. For each potential “Hardy Cell” candidate, the cell-substrateinteraction was evaluated for the following extra-cellular matrices(TABLE 2):

TABLE 2 EXTRA CELLULAR MATRIX Type Source Primaria cultureware syntheticCollagen Type IV human placenta Cell-Tak mussel Fibronectin humanplacenta Poly-D-lysine synthetic Laminin synthetic Elastin bovine Tissueculture plastic control

The optimal ECM was used to coat the scaffold to which the cells willultimately adhere inside the device. The optimal combination of “Hardycell” candidate, ECM, and scaffold were encapsulated using a membranewith intermediate permeability and tested under stringent tissue cultureconditions (aAH or aCSF). Survival was monitored at 1, 2 and 4-weekintervals, using both metabolic and histologic methods.

(c) In vivo device viability screen: Hardy cells from the ECM screenwere further evaluated using different membranes and scaffolds. Theoptimal combinations of cell/ECM/scaffold were encapsulated withdifferent membrane types (membrane with different diffusive properties)and devices were implanted in the rat ventricle. Device performance wasevaluated after a one month in vivo implantation duration.

ARPE-19 viability (as well as other desirable characteristics) wassuperior to other cell lines tested. TABLE 3 summarizes the results forhuman cell lines.

TABLE 3 CELL SCREENING RESULTS - HUMAN CELL LINES Morph- # Cell LineTissue Origin ology Status In Screen  1 C3A* Hepatoblastoma PolygonalViability: passed  2 HepG2* Hepatoblastoma Polygonal Viability: passed 3 HFCH Fetal femur Chondro- Viability: eliminated blast  4 Hs27**Foreskin Fibroblast Viability: passed  5 Hs2-MX1 Foreskin FibroblastViability: eliminated  6 HS683 Glioma Glioblast Viability: eliminated  7A172 Glioblastoma Glioblast Viability: eliminated  8 HS578BST*** Normalbreast Fibroblast Viability: passed  9 184B5 Mammary EpithelialViability: eliminated 10 HCN-1A Cortical Neuron Viability: eliminated 11HCN-2 Cortical Neuron Viability: eliminated 12 HEPM Palate FibroblastMatrix: eliminated 13 NHLF Lung Fibroblast Matrix: eliminated 14 HL60Leukemic Monocyte Viability: eliminated 15 Skin Cri du Chat FibroblastViability: eliminated 16 9.5 Fbr Forebrain Neural Viability: eliminatedstem 17 FHS 74 INT Small intestine Epithelial Viability: eliminated 18MRC-5 Lung Fibroblast Viability: eliminated 19 MRC-9 Fetal lungFibroblast Viability: eliminated 20 WI-38 Lung Fibroblast Viability:eliminated 21 T98G Glioblastoma Glioblast Viability: eliminated 22ARPE-19 Retina Epithelial Viability: passed *These cell lines aretumorigenic **This cell line requires immortalization ***This cell linehas slow growth rate (double every 5 days)

Table 4 summarizes the results for xenogeneic cell lines.

TABLE 4 CELL SCREENING RESULTS - XENOGENEIC CELL LINES Morph- # CellLine Tissue Origin ology Status In Screen  1 SC-1 Rat nerve SchwannViability: eliminated  2 SIRC* Rabbit cornea Fibroblast Viability:passed  3 NCTC 4206 Hamster Fibroblast Viability: eliminated peritoneum 4 Clone 9 Rat liver Epithelial Viability: eliminated  5 TM4 Rat SertoliEpithelial Viability: eliminated  6 PAS Pig embryo brain AstrocyteViability: eliminated  7 BHK Hamster kidney Fibroblast Viability:eliminated  8 CNTF clone 72 Viability: eliminated  9 NT4/5 clone 45Viability: eliminated 10 CHO Hamster ovary Epithelial In vivo:eliminated* 11 3T3 Mouse fibroblast Fibroblast In vivo: eliminated 12C2Cl2 Mouse myoblast Myblast Viability: eliminated 13 B14RAF28-G3Hamster Fibroblast Viability: eliminated peritoneum 14 BRL3A Rat liverEpithelial Viability: eliminated 15 PC12 Rat pheochrom. Pheo- Viability:eliminated chrom. *The cell line is xenogeneic-originated non-human

2. ARPE-19 cells can be genetically modified.

The ARPE-19 cells can be genetically modified to produce a desiredfactor. We transfected ARPE-19 cells with appropriate plasmids usingFugene 6 (a lipid-based transfection reagent from Boehringer Ingelheim)according to the manufacturer's protocol. Polyclonal stable cell lineswere selected, for example, using G418 (Gibco-BRL, Gaithersburg, Md.) ata concentration of 1.0 mg/ml and maintained at 0.25 mg/ml. StableARPE-19/GDNF, ARPE-19/CNTF and ARPE-19/EGFP cell lines were established.GDNF or CNTF output, as measured by ELISA, was between 100-200ng/million cells/24 hr(ng/M/d). (This is one example of a“therapeutically effective” amount.)

(a) CNTF. ARPE-19 cells were transfected with various CNTF-encodingplasmids (see, TABLE 5). ARPE-19 cells transfected with P544(pNUT-IgSP-CNTF (genomic)) were designated NTC201 (ARPE/CNTF; ARPE-P444;CNCM Registry No: I-2524).

TABLE 5 CNTF TRANSFECTION PcDNA3.1. pPI pNUT IgSP-CNTF (hCMV)** (mPGK)**(mMT-1)** cDNA P566* P565* — Genomic — — P544 *verified by restrictionanalysis only **Promoters: hCMV, human cytomegalovirus: mPGK, mousephosphoglycerate kinase; mMT-1, mouse metallothionine.

(b) GDNF. ARPE-19 cells were transfected with five plasmid constructsexpressing hGDNF under different promoters and signal sequences (TABLE6). Cells were transfected by FuGene 6. Transfected cells were selectedby resistance to the drug G418 (Geneticin) or methotrexate.

TABLE 6 PLASMIDS EXPRESSING hGDNF UNDER DIFFERENT PROMOTERS AND SIGNALSEQUENCES Ampli- Pro- Signal Selection fication Plasmid Vector cDNAmoter Peptide Marker Marker p142 pcDNA3 hGDNF CMV HGDNF neomycin p168pcDNA3 hGDNF CMV Ig neomycin p558 pPI-DN hGDNF pgk Ig neomycin DHFR p559pPI-DN hGDNF pgk HGDNF neomycin DHFR p560 pNUT hGDNF MT-1 HGDNF DHFRDHFR CMV = cytomegalovirus; PGK = phoshoglycerate kinase; MT =metallothionein; Ig = immunoglobulin; hGDNF = human glial cell linederived neurotrophic factor; DHFR = dihydrofolate reductase

Conditioned media from the transfected cells was collected and analyzedby Western Blot for the expression of hGDNF. GDNF is a heparin-binding,disulfide-bonded homodimer that is heterologously glycosylated andmigrates on SDS-PAGE with an apparent molecular weight of 33-45 kDa.Unglycosylated GDNF has an apparent molecular weight of 30 kDa (Lin etal., 63(2) J. Neurochem. 758-768 (1994)). Conditioned media werecollected from parental and transfected ARPE-19 for Western Blotanalysis of hGDNF expression. GDNF was semi-purified from conditionedmedia by Cellufine, a cellulose affinity media (Amicon Matrex) withproperties similar to heparin sepharose. Non-specifically bound proteinswere washed away in 25 mM Hepes/150 mM NaCl pH 7.4 and bound proteinswere eluted in 25 mM Hepes/2 M NaCl, pH 7.4. The eluted proteins wereconcentrated with Pall Filtron microfuge concentrators (MWCO 3 kDa) andbuffer exchanged with 25 mM Hepes to achieve a 200-fold concentration ofconditioned media (CM). Up to 1/20 of the non-reduced, semi-purified CMwere loaded onto a denaturing 10-20% gradient Tricine SDS-PAGE gel andelectroblotted onto Immobilon-P PVDF membranes. Western Blots weredetected by chemiluminescence.

A panel of 5 antibodies (3 polyclonals and 2 monoclonals) were used todetect GDNF production. Chemicon rabbit pAB (AB1454); Promega chickenpAb (G2791); R&D Systems goat pAb (AF212NA); Promega mAb (available onlyin GDNF E, ImmunoAssay Systems G3240/G3520 kit); R&D Systems mAb(MAB212). The antibodies with the least non-specific binding and highestsensitivity were monoconals from Promega and R&D Systems. NTC200p559(ARPE/GDNF; ARPE-19/p559; CNCM Registry No: I-2523).

(c) Green fluorescent protein. ARPE-19 cells were transfected with theGreen Fluorescent Protein (GFP) encoding plasmid pEGFP-N1 (Clontech,Palo Alto, Calif.). Since expression of Green Fluorescent Protein can beeasily monitored visually using a fluorescent microscope, the stabilityof the heterologous expression of this foreign gene can be monitoredthroughout many generations. Briefly, ARPE-19 cells were geneticallymodified to express Green Fluorescent Protein. Clonal lines were derivedfrom a polyclonal ARPE-19/GFP by limiting dilution, then expanded to aT-25 flask. One of these lines, NTC200p393(1) (ARPE/GFP; P-393-1; CNCMRegistry No: I-2522) was maintained and passed weekly.

P393-1 was passed at least 5 times since having been expanded to theinitial T-25. P393-1 had undergone over 30 doublings since being cloned.Green Fluorescent Protein expression continued to be robust throughoutthe lines entire lifespan.

3. ARPF-19 has a long life span.

The growth rate of ARPE-19 cells in serum culture medium (DMEM/F12+10%FBS) is approximately 1 doubling/48 hr. The total passage numberexceeded 100. The ARPE-19 cells appear to have a normal phenotype andgrow at a stable rate.

4. ARPE-19 is of human origin.

ARPE-19 is a spontaneously arising retinal pigment epithelia (RPE) cellline derived in 1986 from the normal eyes of a 19-year-old human malewho died from head trauma in a motor vehicle accident (ATCC).

5. ARPE-19 exhibits acceptable in vivo device viability.

The device viability of encapsulated ARPE-19 cells was evaluated in vivoin the central nervous system and eye environments. ARPE-19 cells orARPE-P544 cells (expressing CNTF) were encapsulated and the resultingdevices were implanted into rat ventricle (ICV), rabbit eye, dog eye,and sheep intrathecal space. Briefly, the cells were encapsulated witheither CytoPES 14 (polyethersulfone) or CytoPES 1000 membranes. ForCytoPES14 membranes, the matrix scaffold was PET yarn, 6 strands; theextra-cellular matrix (ECM) coating was Laminin/Collagen IV; the deviceanchor was a titanium loop; the total device length was 1.1 cm; the cellload density was 66 K/μl, the load volume was 6 μl, the holding mediawas Ultra Culture. For CytoPES 1000 membranes, the matrix was PET yarncoated in a two step coating process with human collagen type IV andlaminin at 1 mg/ml and 0.1 mg/ml respectively. For rat ICV, the deviceswere 0.7 cm in length. For dog and rabbit eye, the devices were 1.0 cmin length. For sheep intrathecal space, the devices were 7.0 cm inlength. All devices were non-strutted and E-Beam (electron-beam)sterilized at Titan Systems (Denver, Colo.). The hold media wereUltraculture+1% L-Glutamine or ACSF+0.83% FBS, 0.26% L-Glutamine andbuffer with 15 mM HEPES.

One month after surgical implantation, the devices were explanted andcell viability in the devices was evaluated histologically. The celldevice viability was excellent in all implantation sites examined. Theresults are presented in TABLE 7.

TABLE 7 ARPE-P544 CELL DEVICE VIABILITY AFTER 1 MONTH IN VIVO AnimalDevice model Device Design Implantation site viability Rat 0.7 cm inlength ICV Viable Rabbit 1.0 cm in length Intra-vitreous Viable Dog 1.0cm in length Intra-vitreous Viable Sheep 7.0 cm in length IntrathecalViable

6. ARPE-19 can be modified to deliver an efficacious quantity of growthfactor: Proof of concept.

The therapeutic effect (retinal protection) of CNTF secreting ARPE-P544cells was evaluated in both rat and dog animal models for retinaldegeneration.

(a) Transgenic rat test—unencapsulated ARPE-544. At postnatal day (PD)9, approximately 10⁵ ARPE-P544 cells in 2 μl phosphate buffered saline(PBS) were injected into the vitreous of the left eye of S334ter-3 ratsusing 32 gauge needles. Control eyes (right eyes) were injected withun-transfected ARPE-19 cells. For the CNTF bolus injections, 1 μg CNTFin 1 μl of PBS was injected into the vitreous at PD 9. The eyes werecollected at PD 20, and processed for histologic evaluation. Plasticembedded sections of 1 μm thickness were examined by light microscopy.

In the untreated S334ter transgenic rats, severe photoreceptordegeneration was observed by post delivery (PD) day 20. The outer nucleilayer (ONL) had only one row of nuclei. In the ARPE-P544 injected eyes,the ONL had 5-6 rows of nuclei, while in the control eyes injected withARPE-19 cells, there were 1-2 rows of nuclei remaining. In animalstreated with a bolus injection of purified human recombinant CNTF, theONL had 2-3 rows of nuclei. The results are presented in FIG. 1.

A sustained release of CNTF from the unencapsulated ARPE-19 cellstransfected with CNTF achieved better protection of photoreceptors thana bolus injection of purified CNTF protein.

(b) Mutant dog test—encapsulated ARPE-544. This study was conducted inrod-cone dysplasia (rcd 1) dogs with the rod cyclic GMPphosphodiesterase beta subunit gene (PDE6B) mutation. One set of testsused 6 dogs at 7 weeks of age. Two dogs were affected (retinaldegeneration) and 4 were gene carriers (normal). For the two affecteddogs, the left eye received an ARPE-P544 containing device and the righteye was untreated (control). For the 4 normal dogs, both eyes receivedARPE-P544 containing devices. The devices were 1.1 cm in lengthincluding a titanium anchoring loop. Each device consisted of theCytoPES14 membrane and a PET yarn scaffold coated with laminin andcollagen type IV. The duration of the study was 7 weeks. All devicesexcept two in one normal dog were explanted and evaluated for CNTFoutput by ELISA (R&D Systems) and for cell viability by histologicalanalysis.

The CNTF output for ARPE-P544 was estimated at 150-200 ng/10⁶ cells/24hr (un-encapsulated, in vitro, in complete growth media). The cells weregrown in T-75 flasks and maintained in a DMEM-based medium supplementedwith 10% FBS, in a 5% CO₂, 95% humidity, 37° C. incubator. Prior toencapsulation, the CNTF output of the cells was assayed by an enzymelinked immunosorbent assay (ELISA).

Prior to encapsulation, the devices were assembled in a controlledenvironment, packaged and then e-beam sterilized (see below for detailsof other encapsulation protocols). The ARPE-19 cells were harvested onthe day of encapsulation, and suspended in Ultraculture serum-free mediaat a density of 66,000 cells/μl. The sterile devices were then loadedusing a Hamilton syringe with a volume of 6 μl each. After the infusionof the cellular suspension, the final seal was applied. The cell-loadeddevices were maintained in Ultraculture supplemented with 1%L-glutamine. After 7 days, all devices were assayed for CNTF output byELISA. In vitro cohorts of devices were maintained in six well platesthroughout the course of study. Feeding was performed once a week.

For surgery, animal was sedated with ketamine and xylazine. Body weightand temperature were measured and pupils were dilated with two dropseach of Mydfrin (2.5%) and Cyclogyl (1%). Blood samples were drawn andan intravenous (IV) line established. Anesthesia was induced with sodiumpentothal. The animal was then endotracheally intubated and moved intothe operating room where inhalation of isoflurane was established, andvital signs monitored. Alternatively, inhalation of isoflurane wasestablished before intubation, obviating the need for sedatives andpentothal induction. The animal was then positioned on the operatingtable in the lateral decubitus position and covered with a heating pad.

In the eye implantation procedure, the lids and periorbital area of theeyes were scrub-prepped with dilute iodophor solution, and steriledrapes were secured. The eyelids were retracted with a lid speculum andthe eye positioned with a single conjunctival stitch placed at thelimbus and clamped to the drapes, rotating the eye medially. Undermicroscopic visualization, a 6 mm incision was made through bothconjunctiva and tenons approximately 3.5 mm from the limbus using ablunt scissors. Using a #75 blade a 2.0-3.0 mm longitudinal incision wasmade through the sclera, approximately 4 mm lateral to the limbus toimplant the device through the pars plana. A device was removed from itspackage, rinsed with sterile saline, and examined for gross defects.Using a jewler's forceps, the device was grasped by the tether loop andinserted through the incision into the vitreous. After placement, thesclera was closed with interrupted 8-0 nylon sutures which passedthrough the loop at the external end of the device. The conjunctiva wasclosed with interrupted 8-0 nylon as well. The speculum and drapes wereremoved, and the animal repositioned so the above procedure could berepeated on the contralateral eye.

On the day of surgery, the dogs (7 weeks old, weight approx. 10 lb.)were monitored for heart and respiratory rates, rectal temperature,blood pressure and general body condition. Each animal was identifiedwith a unique number which was noted on the animal. Cyclosporin A(Sandoz, 100 mg/ml, 10 mg/kg) was given once daily (oral), starting oneday prior to implantation. Prednisone (5 mg/kg) and Clavamox (15 mg/kg)were given twice daily (oral), starting at day of implantation. Theanimals were placed on appropriate feed and water ad libitum and werehoused indoors with a dark/light cycle consistent with Eastern StandardTime. Room Temperature was maintained at 18-29° C. (65-84° F.). Humidityrange was maintained at 30-70%.

All animals were monitored daily following the surgical procedure forsigns of surgically-related complications. Animals were monitored on aweekly basis for the presence of the following: coughing, signs ofweight loss, fever, aphthous stomatitis, ocular inflammation ordischarge, or gross motor deficiencies. Animals were weighed andtemperatures taken at pre-implantation within 3 days of implant, and 7days post-implantation. Blood pressures were also taken pre-implantationand within 1 week of sacrifice.

For the collection of blood samples, 30 ml of blood was drawn forimmunology screening at (1) the time of implant and (2) at sacrifice.Ten ml was placed in a heparinized green top tube at room temperaturefor peripheral blood lymphocytes; 10 ml was placed in a red top tube,without anticoagulants, on ice for serum; and 10 ml was submitted for ablood chemistry profile.

At the conclusion of the experiment, the animals were euthanized with anoverdose of pentobarbital 360 mg/kg IV after initial sedation withketamine and pentothal. The devices were surgically explanted, assayedfor CNTF release, and then placed in 4% paraformaldehyde for histologysubmission. The eyes were enucleated and fixed in 50 cc Bouin's solutionat room temperature for 8-12 hr. The eyes were then rinsed in water andstored in 70% EtOH, and submitted for histological examination. Forhistological analysis, 3 slides of paraffin embedded eyes, sectionedthrough optic nerve, dorsal-lateral, stained with hematoxylin and eosin(H&E), were ordered for each eye. The devices were fixed in 4% Paraformaldehyde for 30 min to 2 hr and processed for paraffin orglycidylmethacrylate (GMA) embedding, sectioned, and stained forhistological evaluation. Each device was reviewed to determine celldensity and cell viability.

The CNTF output of devices was evaluated after explantation. Briefly,devices were incubated in Ultraculture (0. 5 ml/well) for 24 hr. whichwas then assayed for CNTF output. The average device output of CNTF was1.62±0.4 ng/device/24 hr. (This is one example of a “therapeuticallyeffective” amount.) The individual device output is presented in TABLE8.

TABLE 8 CNTF OUTPUT FROM EXPLANTED DEVICES CNTF Device # (ng/Device/24hr) 1  1.65 2 1.9 3  2.45 4 1.1 5 1.4 6 1.3 7 1.7 8  1.62 Mean ± SD 1.62± 0.4

Encapsulated ARPE-P544 cells protected photoreceptors in the mutant dogmodel for retinal degeneration. Protection was defined by the degree ofphotoreceptor sparing. The non-affected dogs had 10-12 layers of ONL,while affected, non-treated dogs had 2-3 layers of ONL. The affectedCNTF treated dogs had 5-6 layers of ONL. The results are presented inTABLE 9.

TABLE 9 NUMBER OF NUCLEI IN THE OUTER NUCLEAR LAYER (ONL) IN CNTFTREATED/UNTREATED rcd1 AND NON-AFFECTED DOGS ACROSS 6 AREAS OF THERETINA Superior Retina^(c) Inferior Retina^(d) Dog#, Eye S1 S2 S3 I1 I2I3 1485L^(a) 4-6 6 6 4  4 4 1485R^(a) 3-4 3 4 2-3 2-3 2-3 1489L^(a) 5-67-8 7-8 5-6 4-5 5 1489R^(a) 3-4 3-4 3-4 3-4 4-5 2-3 1487L^(b) 10-1111-12  9-10 11-12 10-11 8 1487R^(b) 11-12 10-11 9 11-12 10 9 ^(a).affected rcd1 dog ^(b). non-affected dog ^(c). Superior Retina = 7-8 10xfields long; 18 40x fields S1 = 2 10x fields peripheral from optic nerve(4-40x fields peripheral)  S2 = midpoint between optic nerve & oraserrata  S3 = 2 10x fields central from ora serrata (4-40x fieldscentral) ^(d). Inferior Retina = 5-6 10x fields long; 10 40x fields  I1= 1 10x fields peripheral from optic nerve (3 4x fields peripheral)  I2= midpoint between optic nerve & ora serrata  I3 = 1 10x fields centralfrom ora serrata (3 40x fields central)

Histological evaluation indicated that all devices contain healthyviable cells. No cellular necrosis was observed in any of the devices.No immune reaction, inflammation or damage to the retina was observed.

7. ARPE-19 triggers low level host immune reaction.

(a) HLA class I and class II-DR. Baseline studies on cell lines Hs27 andARPE-19 were carried out to determine the resting expression levels ofthe human HLA class I and HLA class II-DR molecules. The majorhistocompatibility marker W6/32 is normally expressed on any nucleatedcell. In contrast, the HLA-class II DR marker is expressed on a morespecific population of cells. Normally, the HLA class II DR molecule isfound on B lymphocytes, monocytes, activated T cells, activated naturalkiller (NK) cells, and human progenitor cells.

TABLE 10 summarizes the non activated levels of expression from stainingusing the flow cytometer.

TABLE 10 CELL SCREENING FLOW CYTOMETRY (% POSITIVE) HLA Class II HLAClass I DR Becton Monoclonal Dickinson clone Cell W6/32 IgG 2a L243 IgG2a Hs27 P9 human skin fibroblast 80.1 3.1 ARPE P37 98.1 3.2 Cyno B083110492 positive control 98.7 23.1  BHK NGF P47 negative control  0.1 0.03

(b) Allogeneic vs. xenogeneic antibody responses. Serum samples fromeither dog hosts or human hosts were evaluated using ARPE-19, Hs27(human derived) and SIRC (rabbit derived) as target cells. To evaluatethe specific anti-ARPE-19 IgG titer, ARPE-19 cells were used as targetcells for flow cytometric analysis. ARPE-19 cells (100,000 cells/100 μl)were incubated with 25 μl of host serum, serially diluted up to a titerof 4×10⁶ and were then tagged with a fluorescein isothiocyanate (FITC)labeled second antibody (ICN Pharmaceuticals, Costa Mesa, Calif., USA).Data acquisition and histogram analysis was performed using a BectonDickinson FACSort™(Becton Dickinson Immunocytometry Systems, San Jose,Calif., USA) with a 488 nm excitation and a single air cooled argonlaser. Data were interpreted using CELLQuest™(Becton DickinsonImmunocytometry Systems, San Jose, Calif., USA) software with a 256channel program and geometric/channel preferences.

All data were reported as an increase in mean channel shift fluorescence(MCS) over the negative control samples. A shift of greater than 10channels was considered positive. The results show that dog serumcontains antibodies at much higher titer compared to human serum (FIG.2).

Allogeneic vs. xenogeneic complement dependent cytotoxicity (CDC)responses. To determine the titer of cytotoxic antibodies specific toARPE-19 cells, ARPE-19 cells were used as target cells in acomplement-dependent cytotoxicity assay. The complement fixation ofcytotoxic antibody titer was evaluated using either dog serum or humanserum samples. ARPE-19, Hs27 and SIRC cells were used as the targets.All serum samples were set up in two groups of triplicates and assayedemploying a standard National Institutes of Health (NIH) tissue typingtechnique (American Society for Histocompatibility and Immunogenetics(ASHI) Manual, 1994). One group of triplicates was analyzed with 1 μl ofhost serum and ARPE-19 cells (1000 cells/1 μl); the second group oftriplicates was assayed in the same manner with the addition of 5 μlexogenous prescreened rabbit complement (Pel Freez Brown Deer, Wis.,USA). Samples were prepared using a two-color immunofluorescentmicrocytotoxic analysis procedure. A 1 hr room temperature incubation ofhost serum and ARPE-19 cells was followed by a second 1 hr roomtemperature incubation with or without additional rabbit complement. Inthe same microtiter well, the percentage of living cells (negativereactivity) was visualized using fluorescein diacetate, while thepercentage of dead cells (positive reactivity) was visualized usingpropidium iodide. All serum specimens were set up in 1:2 serialdilutions to a maximum of a 1:100,000 dilution. All data was scoredusing a Nikon Diaphot™ inverted fluorescence microscope with a 488 nmwavelength excitation.

The results show that dog serum contained antibodies which can activatecomplement in the presence of these cells. In contrast, human serum didnot fix the complement (FIG. 3).

8. The ARPE-19 cell line is nontumorigenic.

A tumorigenicity study of ARPE-19 cells was conducted following theprocedure required by the U.S. Food and Drug Administration (FDA),Points to consider in the characterization of cell lines used in theproductions of biologicals, 58 Federal Register 42974 (Aug. 12, 1993).Briefly, 10 million cells in 0.2 ml of serum free medium (PBS+10mM.glucose) were injected subcutaneously into each nude mouse(irradiated Swiss nude mice, 2-7 days after irradiation, n=10). Theanimals were observed daily for 3 weeks for the evidence of tumorformation. At that time, half the animals were sacrificed, dissected andhistologically examined to ensure the presence of the cells. Theremaining animals were observed for an additional 12 weeks. The ARPE-19cells showed no tumor formation in the nude mice for the 15-week studyperiod.

B. Cell Encapsulation Methods and Devices

Encapsulated cell therapy is based on the concept of isolating cellsfrom the recipient host's immune system by surrounding the cells with asemipermeable biocompatible material before implantation within thehost. The invention includes a device in which ARPE-19 cells areencapsulated in an immunoisolatory capsule. An “immunoisolatory capsule”means that the capsule, upon implantation into a recipient host,minimizes the deleterious effects of the host's immune system on theARPE-19 cells in the core of the device. ARPE-19 cells areimmunoisolated from the host by enclosing them within implantablepolymeric capsules formed by a microporous membrane. This approachprevents the cell-to cell contact between host and implanted tissues,eliminating antigen recognition through direct presentation. Themembranes used can also be tailored to control the diffusion ofmolecules, such as antibody and complement, based on their molecularweight (Lysaght et al., 56 J. Cell Biochem. 196 (1996), Colton, 14Trends Biotechnol. 158 (1996)). Using encapsulation techniques, ARPE-19cells can be transplanted into a host without immune rejection, eitherwith or without use of immunosuppressive drugs. Useful biocompatiblepolymer capsules usually contain a core that contains cells, eithersuspended in a liquid medium or immobilized within an immobilizingmatrix, and a surrounding or peripheral region of permselective matrixor membrane (“jacket”) that does not contain isolated cells, that isbiocompatible, and that is sufficient to protect cells in the core fromdetrimental immunological attack. Encapsulation hinders elements of theimmune system from entering the capsule, thereby protecting theencapsulated ARPE-19 cells from immune destruction. The semipermeablenature of the capsule membrane also permits the biologically activemolecule of interest to easily diffuse from the capsule into thesurrounding host tissue.

The capsule can be made from a biocompatible material. A “biocompatiblematerial” is a material that, after implantation in a host, does notelicit a detrimental host response sufficient to result in the rejectionof the capsule or to render it inoperable, for example throughdegradation. The biocompatible material is relatively impermeable tolarge molecules, such as components of the host's immune system, but ispermeable to small molecules, such as insulin, growth factors, andnutrients, while allowing metabolic waste to be removed. A variety ofbiocompatible materials are suitable for delivery of growth factors bythe composition of the invention. Numerous biocompatible materials areknown, having various outer surface morphologies and other mechanicaland structural characteristics. Preferably the capsule of this inventionwill be similar to those described by PCT International patentapplications WO 92/19195 or WO 95/05452, incorporated by reference; orU.S. Pat. Nos. 5,639,275; 5,653,975; 4,892,538; 5,156,844; 5,283,187; orU.S. Pat. No. 5,550,050, incorporated by reference. Such capsules allowfor the passage of metabolites, nutrients and therapeutic substanceswhile minimizing the detrimental effects of the host immune system.Components of the biocompatible material may include a surroundingsemipermeable membrane and the internal cell-supporting scaffolding.Preferably, the transformed cells are seeded onto the scaffolding, whichis encapsulated by the permselective membrane. The filamentouscell-supporting scaffold may be made from any biocompatible materialselected from the group consisting of acrylic, polyester, polyethylene,polypropylene polyacetonitrile, polyethylene teraphthalate, nylon,polyamides, polyurethanes, polybutester, silk, cotton, chitin, carbon,or biocompatible metals. Also, bonded fiber structures can be used forcell implantation (U.S. Pat. No. 5,512,600, incorporated by reference).Biodegradable polymers include those comprised of poly(lactic acid) PLA,poly(lactic-coglycolic acid) PLGA, and poly(glycolic acid) PGA and theirequivalents. Foam scaffolds have been used to provide surfaces ontowhich transplanted cells may adhere (PCT International patentapplication Ser. No. 98/05304, incorporated by reference). Woven meshtubes have been used as vascular grafts (PCT International patentapplication WO 99/52573, incorporated by reference). Additionally, thecore can be composed of an immobilizing matrix formed from a hydrogel,which stabilizes the position of the cells. A hydrogel is a3-dimensional network of cross-linked hydrophilic polymers in the formof a gel, substantially composed of water.

Various polymers and polymer blends can be used to manufacture thesurrounding semipermeable membrane, including polyacrylates (includingacrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers,polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulosenitrates, polysulfones (including polyether sulfones), polyphosphazenes,polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well asderivatives, copolymers and mixtures thereof. Preferably, thesurrounding semipermeable membrane is a biocompatible semipermeablehollow fiber membrane. Such membranes, and methods of making them aredisclosed by U.S. Pat. Nos. 5,284,761 and 5,158,881, incorporated byreference. The surrounding semipermeable membrane is formed from apolyether sulfone hollow fiber, such as those described by U.S. Pat. No.4,976,859 or U.S. Pat. No. 4,968,733, incorporated by reference. Analternate surrounding semipermeable membrane material ispoly(acrylonitrile/covinyl chloride).

The capsule can be any configuration appropriate for maintainingbiological activity and providing access for delivery of the product orfunction, including for example, cylindrical, rectangular, disk-shaped,patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule canbe coiled or wrapped into a mesh-like or nested structure. If thecapsule is to be retrieved after it is implanted, configurations whichtend to lead to migration of the capsules from the site of implantation,such as spherical capsules small enough to travel in the recipienthost's blood vessels, are not preferred. Certain shapes, such asrectangles, patches, disks, cylinders, and flat sheets offer greaterstructural integrity and are preferable where retrieval is desired.

When macrocapsules are used, preferably between 10³ and 10⁸ ARPE-19cells are encapsulated, most preferably 10⁵ to 10⁷ ARPE-19 cells areencapsulated in each device. Dosage may be controlled by implanting afewer or greater number of capsules, preferably between 1 and 10capsules per patient.

The scaffolding may be coated with extracellular matrix (ECM) molecules.Suitable examples of extracellular matrix molecules include, forexample, collagen, laminin, and fibronectin. The surface of thescaffolding may also be modified by treating with plasma irradiation toimpart charge to enhance adhesion of ARPE-19 cells.

Any suitable method of sealing the capsules may be used, including theuse of polymer adhesives or crimping, knotting and heat sealing. Inaddition, any suitable “dry” sealing method can also be used, asdescribed, e.g., in U.S. Pat. No. 5,653,687, incorporated by reference.

The encapsulated cell devices are implanted according to knowntechniques. Many implantation sites are contemplated for the devices andmethods of this invention. These implantation sites include, but are notlimited to, the central nervous system, including the brain, spinal cord(see, U.S. Pat. Nos. 5,106,627, 5,156,844, and 5,554,148, incorporatedby reference), and the aqueous and vitreous humors of the eye (see, PCTInternational patent application WO 97/34586, incorporated byreference).

C. Genetic Engineering of ARPE-19 Cells

As described above, ARPE-19 cells can be genetically engineered. Theterms “genetic modification” and “genetic engineering” refer to thestable or transient alteration of the genotype of an APRE-19 cell byintentional introduction of exogenous DNA. DNA may be synthetic, ornaturally derived, and may contain genes, portions of genes, or otheruseful DNA sequences. The term “genetic modification” is not meant toinclude naturally occurring alterations such as that which occursthrough natural viral activity, natural genetic recombination, or thelike.

Any useful genetic modification of the APRE-19 cells is within the scopeof the invention. For example, APRE-19 cells may be modified to produceor increase production of a biologically active substance such as aneurotransmitter or growth factor or the like. The genetic modificationcan be performed either by infection with viral vectors (retrovirus,modified herpes viral, herpes-viral, adenovirus, adeno-associated virus,and the like) or transfection using methods known in the art(lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like) (see, Maniatis et al., in MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y.,1982)). For example, the chimeric gene constructs can contain viral, forexample retroviral long terminal repeat (LTR), simian virus 40 (SV40),cytomegalovirus (CMV); or mammalian cell-specific promoters. Inaddition, the vectors can include a drug selection marker, such as theE. coli aminoglycoside phosphotransferase gene, which when co-infectedwith the test gene, confers resistance to geneticin (G418), a proteinsynthesis inhibitor.

APRE-19 cells can be genetically modified using transfection withexpression vectors. An “expression vector” is a nucleic acid eitherintegrated in the genome or present in the cytoplasm, and capable ofpermitting the expression of the polypeptide, protein or viral vector.In one protocol, vector DNA containing the genes are diluted in 0.1×TE(1 mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of 40 μg/ml. 22 μl ofthe DNA is added to 250 μl of 2×HBS (280 mM NaCl, 10 mM KCl, 1.5 mMNa₂HPO₄, 12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5 mlplastic tube. 31 μl of 2 M CaCl₂ is added slowly and the mixture isincubated for 30 minutes (min) at room temperature. During this 30 minincubation, the cells are centrifuged at 800 g for 5 min at 4° C. Thecells are resuspended in 20 volumes of ice-cold PBS and divided intoaliquots of 1×10⁷ cells, which are again centrifuged. Each aliquot ofcells is resuspended in 1 ml of the DNA-CaCl₂ suspension, and incubatedfor 20 min at room temperature. The cells are then diluted in growthmedium and incubated for 6-24 hr at 37° C. in 5%-7% CO₂. The cells areagain centrifuged, washed in PBS and returned to 10 ml of growth mediumfor 48 hr.

Suitable vehicles for direct DNA, plasmid polynucleotide, or recombinantvector administration include, without limitation, saline, or sucrose,protamine, polybrene, polylysine, polycations, proteins, calciumphosphate, or spermidine. See e.g, PCT International patent applicationWO 94/01139.

APRE-19 cells can also be genetically modified using calcium phosphatetransfection techniques. For standard calcium phosphate transfection,the cells are mechanically dissociated into a single cell suspension andplated on tissue culture-treated dishes at 50% confluence (50,000-75,000cells/cm²) and allowed to attach overnight. In one protocol, themodified calcium phosphate transfection procedure is performed asfollows: DNA (15-25 μg) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA,pH 7.5) diluted to 440 μλ with TE, and 60 μl of 2 M CaCl₂ (pH to 5.8with 1M HEPES buffer) is added to the DNA/TE buffer. A total of 500 μlof 2×HeBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na₂HPO₄, mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is added dropwiseto this mix. The mixture is allowed to stand at room temperature for 20min. The cells are washed briefly with 1×HeBS and 1 ml of the calciumphosphate precipitated DNA solution is added to each plate, and thecells are incubated at 37° C. for 20 min. Following this incubation, 10ml of “Complete Medium” is added to the cells, and the plates are placedin an incubator (37° C., 9.5% CO₂) for an additional 3-6 hours. The DNAand the medium are removed by aspiration at the end of the incubationperiod. The cells are washed, fresh medium is added and then cells arereturned to the incubator.

Alternatively, the calcium phosphate co-precipitation technique can beused, as described in PCT International patent application WO 93/06222.

Moreover, ARPE-19 cells can be genetically engineered to produce adesired secreted factor. The desired secreted factor can be encoded byeither a synthetic or recombinant polynucleotide. The term “recombinant”refers to the molecular biological technology for combiningpolynucleotides to produce useful biological products, and to thepolynucleotides and peptides produced by this technology. Thepolynucleotide can be a recombinant construct (such as a vector orplasmid) which contains the polynucleotide encoding the desired secretedfactor under the operative control of polynucleotides encodingregulatory elements such as promoters, termination signals, and thelike. “Operatively linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence operatively linkedto a coding sequence is ligated such that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Control sequence” refers to polynucleotide sequences whichare necessary to effect the expression of coding and non-codingsequences to which they are ligated. Control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence. In addition, “control sequences” refers to sequences whichcontrol the processing of the peptide encoded within the codingsequence; these can include, but are not limited to sequencescontrolling secretion, protease cleavage, and glycosylation of thepeptide. The term “control sequences” is intended to include, at aminimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. A “codingsequence” is a polynucleotide sequence which is transcribed andtranslated into a polypeptide. Two coding polynucleotides are “operablylinked” if the linkage results in a continuously translatable sequencewithout alteration or interruption of the triplet reading frame. Apolynucleotide is operably linked to a gene expression element if thelinkage results in the proper function of that gene expression elementto result in expression of the desired secreted factor. “Transformation”is the insertion of an exogenous polynucleotide (i.e., a “transgene”)into a host cell. The exogenous polynucleotide is integrated within thehost genome. A polynucleotide is “capable of expressing” a desiredsecreted factor if it contains nucleotide sequences which containtranscriptional and translational regulatory information and suchsequences are “operably linked” to polynucleotide which encode thedesired secreted factor. A polynucleotide that encodes a peptide codingregion can be then amplified, for example, by preparation in a bacterialvector, according to conventional methods, for example, described in thestandard work Sambrook et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Press 1989). Expression vehicles include plasmids orother vectors.

The polynucleotide encoding the desired secreted factor can be preparedby chemical synthesis methods or by recombinant techniques. Thepolypeptides can be prepared conventionally by chemical synthesistechniques, such as described by Merrifield, 85 J. Amer. Chem. Soc.2149-2154 (1963) (see, Stemmer et al, 164 Gene 49 (1995)). Syntheticgenes, the in vitro or in vivo transcription and translation of whichwill result in the production of the desired secreted factor protein canbe constructed by techniques well known in the art (see Brown et al., 68Methods in Enzymology 109-151 (1979)). The coding polynucleotide can begenerated using conventional DNA synthesizing apparatus such as theApplied Biosystems Model 380A or 380B DNA synthesizers (commerciallyavailable from Applied Biosystems, Inc., 850 Lincoln Center Drive,Foster City, Calif., USA).

Polynucleotide gene expression elements useful for the expression ofcDNA encoding desired secreted factor include, but are not limited to(a) viral transcription promoters and their enhancer elements, such asthe SV40 early promoter, Rous sarcoma virus LTR, and Moloney murineleukemia virus LTR; (b) splice regions and polyadenylation sites such asthose derived from the SV40 late region; and (c) polyadenylation sitessuch as in SV40. Recipient cells capable of expressing the desiredsecreted factor are then transfected. The transfected recipient cellsare cultured under conditions that permit expression of the desiredsecreted factor, which is recovered from the culture. ARPE-19 cells canbe used in connection with poxvirus vectors, such as vaccinia orswinepox. Suitable non-pathogenic viruses which can be engineered tocarry the synthetic gene into the cells of the host include poxviruses,such as vaccinia, adenovirus, retroviruses and the like. A number ofsuch non-pathogenic viruses are commonly used for human gene therapy,and as carrier for other vaccine agents, and are known and selectable byone of skill in the art. The selection of other suitable host cells andmethods for transformation, culture, amplification, screening andproduct production and purification can be performed by one of skill inthe art by reference to known techniques (see, e.g., Gething & Sambrook,293 Nature 620-625 (1981)). Another preferred system includes thebaculovirus expression system and vectors.

The polynucleotide encoding the desired secreted factor can be used in avariety of ways. For example, a polynucleotide can express the desiredsecreted factor peptide in vitro in a host cell culture. The expresseddesired secreted factor, after suitable purification, can then beincorporated into a pharmaceutical reagent or vaccine (described below).

Determinations of the sequences for the polynucleotide coding regionthat codes for the desired secreted factor described herein can beperformed using commercially available computer programs, such as DNAStrider and Wisconsin GCG. Owing to the natural degeneracy of thegenetic code, the skilled artisan will recognize that a sizable yetdefinite number of DNA sequences can be constructed which encode theclaimed peptides (see, Watson et al., Molecular Biology of the Gene,436-437 (the Benjamin/Cummings Publishing Co. 1987)).

The term “biological agent” refers to any agent, such as a virus,protein, peptide, amino acid, lipid, carbohydrate, nucleic acid,nucleotide, drug, pro-drug or other substance that may have an effect onneural cells whether such effect is harmful, beneficial, or otherwise.Biological agents that are beneficial to neural cells are “neurologicalagents”, a term which encompasses any biologically or pharmaceuticallyactive substance that may prove potentially useful for theproliferation, differentiation or functioning of CNS or eye cells ortreatment of neurological or opthalmological disease or disorder. Forexample, the term may encompass certain neurotransmitters,neurotransmitter receptors, growth factors, growth factor receptors, andthe like, as well as enzymes used in the synthesis of these agents.

When the genetic modification is for the production of a biologicalagent, the substance can be one that is useful for the treatment of agiven CNS or eye disorder. APRE-19 cells can be genetically modified toexpress a biologically active agent, such as growth factors, growthfactor receptors, neurotransmitters, neurotransmitter synthesizinggenes, neuropeptides, and chromaffin granule amine transporter. Forexample, it may be desired to genetically modify cells so they secrete aproliferation-inducing growth factor or a differentiation-inducinggrowth factor.

The biological agent can be basic fibroblast growth factor (bFGF), acidfibroblast growth factor, epidermal growth factor, transforming growthfactor α, transforming growth factor β, nerve growth factor, insulinlike growth factor, platelet derived growth factor, glia-derivedneurotrophic factor, brain derived neurotrophic factor, ciliaryneurotrophic factor, phorbol 12-myristate 13-acetate, tryophotin,activin, thyrotropin releasing hormone, interleukins, bone morphogenicprotein, macrophage inflammatory proteins, heparin sulfate,amphiregulin, retinoic acid, tumor necrosis factor α, fibroblast growthfactor receptor, epidermal growth factor receptor, or other agentsexpected to have therapeutically useful effects on potential targettissues. Examples of biological agents include trophic factors such asglial-derived neurotrophic factor (GDNF); regulators of intracellularpathways associated with growth factor activity such as staurosporine,CGP-4 1251, and the like; hormones; various proteins and polypeptidessuch as interleukins; oligonucleotides such as antisense strandsdirected, for example, against transcripts for receptors; heparin-likemolecules; and a variety of other molecules that have an effect onradial glial cells or CNS neural stem cell.

ARPE-19 cells that secrete IL-2 can be created by transfection ofARPE-19 with with the plasmid vector pBCMG-hygro-hIL-2 (Roux et al., 159J. Cell. Physiol. 101-113 (1994)), an episomal expression vectorcontaining the human IL-2 cDNA sequence under the transcriptionalcontrol of a cytomegalovirus (CMV) promoter including a rabbit β-globinintron, followed by a poly(A) sequence, and a hygromycin-resistant genefor selection. To introduce an expression vector encoding the hIL-2protein into the ARPE-19 cell line, the calcium phosphate precipitationtechnique can be used. The pPCHIL plasmid (pBCMG-hIL-2) contains thehIL-2 cDNA sequence followed by the Hygromycin B resistance gene forselection. Cells which have stably integrated foreign DNA into theirgenome are selected in presence of Hygromycin B in the medium.

ARPE-19 cells that secrete IL-10 can be created. Interleukin-10 (IL-10),produced by the Th, subset of CD₄ cells, suppresses cytokine productionby the Th₁, subset of CD4⁺ helper T-lymphocytes. IL-10 also inhibits theproduction of numerous pro-inflammatory cytokines by monocytes. IL-10expression has been detected in human malignant gliomas and at higherlevels in malignant vs. low grade tumors. This has led to the hypothesisthat endogenous IL-10 functions to suppress anti-glioma immunity withinbrain. Despite the potentially immunosuppressive and anti-inflammatoryactions of endogenous IL-10, evidence is mounting that transgenicIL-10produced at high levels by engineered tumor cells can inhibitgrowth of systemic tumors by either stimulating anti-tumor immunity orinhibiting tumor-associated angiogenesis. IL-10-producing ARPE-19 cellscan be created by transfection with the plasmid pBMGneo. IL-10 in thepresence of lipofectamine (GIBCO) using a procedure similar to that ofKundu et al., 88 J. Natl. Cancer Inst. 536-41 (1996).

ARPE-19 cells that secrete FGF can be created. Fibroblast growth factor(FGF) is an endothelial cell mitogen that can be neuroprotective forother cell types within the central nervous system. The ARPE-19 cellline can be genetically altered to express a chimeric human FGF-1 geneconsisting of the hst/KS3 signal sequence of FGF-4 fused in-frame toFGF-1 (sp-hst/KS3:FGF-1) (Forough et al., 268 J. Biol. Chem. 2960-8(1993)).

APRE-19 cells can be engineered to produce various neurotransmitters ortheir receptors such as serotonin, L-dopa, dopamine, norepinephrine,epinephrine, tachykinin, substance P, endorphin, enkephalin, histamine,N-methyl D-aspartate, glycine, glutamate, GABA, ACh, and the like.Useful neurotransmitter-synthesizing genes include TH, DDC, DBH, PNMT,GAD, tryptophan hydroxylase, ChAT, and histidine decarboxylase. Genesthat encode for various neuropeptides, which may prove useful in thetreatment of CNS disorders, include substance-P, neuropeptide-Y,enkephalin, vasopressin, VIP, glucagon, bombesin, CCK, somatostatin,calcitonin gene-related peptide, and the like.

Alternatively, ARPE-19 cells can be constructed to produce retroviralgene transfer vectors using the methods of U.S. Pat. No. 5,614,404,describing recombinant viral vectors which coexpress heterologouspolypeptides capable of assembling into defective nonself-propagatingviral particles. Viruses useful as gene transfer vectors includeretrovirus, which are the vectors most commonly used in human clinicaltrials. To generate a gene therapy vector, the gene of interest iscloned into a replication-defective retroviral plasmid which containstwo long terminal repeats (LTR), a primer binding site, a packagingsignal, and a polypurine tract essential to reverse transcription andthe integration functions of retrovirus after infection. To produceviral vector, the plasmid form of a vector is transfected into apackaging cell line which produces Gag, Pol and Env of the retroviralstructural proteins required for particle assembly. A producer cell lineis usually generated using a selective marker, often a G418 resistantgene carried by the retroviral vector. The resulting cell line can beencapsulated, as described in PCT International patent application WO97/44065, which describes biocompatible capsules containing livingpackaging cells that secrete a viral vector for infection of a targetcell, and methods of delivery for an advantageous infectivity of thetarget cells.

The effects of the biological agents on cells of the CNS or eye in therecipient host can be identified in vitro based upon significantdifferences between model cell cultures for central nervous system cells(such as rat pheochromocytomaPC12 cells, cultured primary centralnervous neurons, etc.); or eye cells (such as the IO/LD7/4 cell line,ARPE-19 cells, cultured retinal pigment epithelial cells, etc.) relativeto control cultures with respect to criteria such as the ratios ofexpressed phenotypes (neurons, glial cells, or neurotransmitters orother markers), cell viability and alterations in gene expression.Physical characteristics of the cells can be analyzed by observing celland neurite morphology and growth with microscopy. The induction ofexpression of new or increased levels of proteins such as enzymes,receptors and other cell surface molecules, or of neurotransmitters,amino acids, neuropeptides and biogenic amines can be analyzed with anytechnique known in the art which can identify the alteration of thelevel of such molecules. These techniques include immunohistochemistryusing antibodies against such molecules, or biochemical analysis. Suchbiochemical analysis includes protein assays, enzymatic assays, receptorbinding assays, enzyme-linked immunosorbant assays (ELISA),electrophoretic analysis, analysis with high performance liquidchromatography (HPLC), Western blots, and radioimmune assays (RIA).Nucleic acid analysis such as Northern blots and PCR can be used toexamine the levels of mRNA coding for these molecules, or for enzymeswhich synthesize these molecules. Also, the cellular detection oftranscripts of the desired secreted factor in vivo can be demonstratedby immunochemistry or by other immunological methods.

D. Therapeutic Usefulness of Polymer Encapsulated ARPE-9 Cell Deliveryof Growth factors

The central nervous system is site that is subject to chronicdegeneration. Growth factors are known to have a tremendous therapeuticpotential for treating neuro-degenerative disorders. For example,polymer-encapsulated xenogeneic cells that have been geneticallyengineered to secrete growth factors can protect against lesion-inducedcell loss in the central nervous system in rats (Winn et al., 91 Proc.Natl. Acad. Sci. USA 2324-8 (1994)), primates (Emerich et al., 349 J.Comp. Neurol. 148-64 (1994)), and aged primates (Kordower et al., 91Proc. Natl. Acad. Sci. USA 10898-902 (1994)). Therapeutic effects havebeen produced with polymer-encapsulated cell devices directly deliveringvarious growth factors to a range of target sites in the central nervoussystem with no evidence of adverse effects (Emerich et al., 130 Exp.Neurol. 141-50 (1994), Emerich et al, 736 Brain Res. 99-110 (1996),Emerich et al., 349 J. Comp. Neurol. 148-64 (1994), Hoffman et al., 122Exp. Neurol. 100-6 (1993), Kordower et al., 72 Neuroscience 63-77(1996), Kordower et al., 91 Proc. Natl. Acad. Sci. USA 10898-902 (1994),Winn et al., 91 Proc. Natl. Acad. Sci. USA 2324-8 (1994)). The safety ofpolymer-encapsulated cell delivery of growth factors is supported bystudies that found no adverse effects in animals receiving growthfactors delivered to the brain for up to one year (Lindner et al., 5Cell Transplant. 205-23 (1996), Winn et al., 140 Exp. Neurol. 126-38(1996)). These studies found no adverse effects even in tests of learnedbehaviors, which are extremely sensitive to neurotoxicity.

The retina is another site subject to chronic degeneration. Developmentof treatments for retinal degeneration is also complicated by problemsof drug delivery to the posterior chamber of the eye. Recently, severalstudies have shown that ciliary neurotrophic factor (CNTF) can betherapeutic for ophthalmic disorders. CNTF has been shown to protect theretina from ischemic injury (Unoki & LaVail, 35 Invest. Ophthalmol. Vis.Sci. 907-15 (1994)). LaVail et al. (89 Proc. Natl. Acad. Sci. USA11249-53 (1992); and 39 Invest. Ophthalmol. Vis. Sci. 592-602 (1998))reported that CNTF exhibited more therapeutic potential than 8 othergrowth factors with respect to the ability of growth factors to protectretinal photoreceptors in rat eyes exposed to light-induceddegeneration. Cayouette et al. (18 J. Neurosci. 9282-93 (1998),Cayouette & Gravel, 8 Hum. Gene Ther. 423-30 (1997)) has shown thatchronic CNTF delivery produces a lasting cell sparing and improves thefunction of surviving photoreceptors in mouse models of retinitispigmentosa.

Age-related macular degeneration (AMD) is the leading cause ofirreversible visual loss in the USA. Age-related macular degeneration isthe most common geriatric eye disorder leading to blindness and ischaracterized by degeneration of the neuroepithelium in the macular areaof the eye. Apolipoprotein E (apoE) appears to be associated withneurodegeneration. The dry form of the disease is more common than thewet, but the wet form causes the most severe vision loss. Other thanvision aids (e.g., glasses, magnifiers), no treatments or preventivemeasures are currently available for patients with dry maculardegeneration, and laser photocoagulation with fluorescein angiography isthe only clinically proven therapy for neovascular disease (see, Starret al., 103(5) Postgrad Med. 153-6, 161-4 (1998)). Laserphotocoagulation of choroidal neovascular membranes (CNVMs) in exudativeAMD has previusly been the only well-studied and widely acceptedtreatment modality. This treatment is beneficial for only a smallminority of patients who show well-demarcated “classic” CNVMs.

Retinitis pigmentosa (RP) is a genetic disorder that causes thedegeneration of cells in the retina. If severe, it may lead to completeblindness.

Diabetic eye disease refers to a group of sight-threatening eye problemsthat people with diabetes may develop as a complication of the disease.They include diabetic retinopathy, which damages blood vessels in theretina, the light-sensitive tissue at the back of the eye thattranslates light into electrical impulses that the brain interprets asvision. Diabetic retinopathy affects about half of the Nation'sestimated 16 million people with diabetes have at least early signs ofdiabetic retinopathy. Of this group, about 700,000 have serious retinaldisease, with approximately 65,000 Americans progressionally each yearto proliferative retinopathy, the disease's most sight-threateningstage. Annually as many as 25,000 people go blind from the disorder,making it a leading cause of blindness among working-age Americans.

The cost of diabetic retinopathy is high, as a year of blindness coststhe U.S. Government approximately $13,607 annually per person in SocialSecurity benefits, 1 income tax revenue, and health care expenditures.

Because growth factors are known to be useful in treating neurologicalor retina degeneration, and because encapsulated ARPE-19 cells can begenetically engineered to secrete growth factors, the invention providesa method for treating neurological or retina degeneration.

E. Conclusion

The ARPE-19 cell line is surprisingly useful for the cell-based deliveryof factors to a recipient host. For example:

(a) The ARPE-19 cell line is a platform cell line for cellular therapy.

(b) Transplantation of ARPE-19 cells is useful for administering adesired therapy factor for treating neurodegenerative diseases.

(c) The ARPE-19 cell line is a platform cell line for cellular therapywhenin the ARPE-19 cells are unencapsulated.

(d) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells are encapsulated.

(e) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells the cells are genetically modified to secretea desired therapeutic factor.

(f) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells are genetically modified to secrete a desiredtherapeutic protein when the desired proteins include (but are notlimited to) neurotrophins, interleukins, cytokines, anti-apoptotic,angiogenic, and anti-angiogenic factors, and antigens. Such factors alsoinclude brain derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4),CNTF, Axokine (second generation ciliary neurotrophic factor (CNTF);RegeneronPharmaceuticals Inc.), basic fibroblast growth factor (bFGF),the insulin like growth factors IGF I and IGF II, TGFβ II, theheparin-binding cytokine Midkine (MK), interleukin 1 (IL-1β), tumornecrosis factor (TNF), nerve growth factor (NGF), IL-2/3, ILF, IL-6,Neurturin (NTN), Neublastin, VEGF, glial cell line-derived neurotrophicfactor (GDNF), platelet-derived growth factor (PDGF), lensepithelium-derived growth factor (LEDGF), and pigment epithelium-derivedfactor (PEDF).

(g) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells are implanted into a mammal, to administer atherapeutically effective amount of desired factor to the mammal.

(h) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells are implanted into the central nervous system,eye or any other tissue of interest.

(i) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells are implanted into the central nervous system,where the central nervous system sites include ventricular andintrathecal spaces, striatum, and other sites in the brain or spinalcord parenchyma.

(j) The ARPE-19 cell line is a platform cell line for cellular therapywherein the ARPE-19 cells are implanted into the eye, where the eyesites include subretinal and intra-vitreal spaces.

(k) Transplantation of ARPE-19 cells is useful for administering atherapeutic protein for treating degenerative diseases, where thedegenerative diseases include (but not limited to) Parkinson's Disease,Huntington's Disease, ALS, Alzheimer's Disease, Spinal Cord Injury,Retinopathy of Prematurity, Diabetic Retinopathy, Age-Related MacularDegeneration, Glaucoma, Retinitis Pigmentosa, Cataract Formation,Retinoblastoma, Retinal Ischemia.

(l) Transplantation of ARPE-19 cells is a method for administering atherapeutic protein for treating cancer and cancer related disorders,cardiovascular deseases, asthma, metabolic diseases and other relevantpathologies.

(m) Transplantation of ARPE-19 cells is a method for administering adesired antigenic factor as a vaccine.

(n) The ARPE-19 cell line can be a packaging cell line to produce viralgene transfer vectors.

(o) Transplantation of ARPE-19 cells is a method for delivering adesired factor to a recipient host. ARPE-19 cells, encapsulated within asemipermeable membrane that allows the diffusion of the growth factor;are implanted into a target region within the recipient host, such thatthe encapsulated ARPE-19 cell secretes the desired factor to the targetregion.

The deposited subject cultures discussed above are deposited underconditions that ensure that access to the cultures will be availableduring the pendency of the patent application disclosing them to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits areavailable as required by foreign patent laws in countries wherecounterparts of the subject application, or its progeny, are filed.However, the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast 30 years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures plus 5 years afterthe last request for a sample from the deposit. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the conditions of thedeposits. All restrictions on availability to the public of the subjectculture deposits will be irrevocably removed upon granting of a patentdisclosing them.

The details of one or more embodiments of the invention are set forth inthe accompanying description above. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. All patents and publications citedin this specification are incorporated by reference.

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto.

We claim:
 1. An implantable cell culture device, the device comprising:(a) a semipermeable membrane permitting the diffusion of a growth factortherethrough; and (b) ARPE-19 cells that are genetically engineered toproduce the growth factor disposed within the semipermeable membrane. 2.The device of claim 1, wherein the semipermeable membrane isimmunoisolatory.
 3. The device of claim 1, wherein the semipermeablemembrane is microporous.
 4. The device of claim 1, wherein thesemipermeable membrane permits the diffusion of a substance oftherapeutic interest.
 5. The device of claim 1, wherein the devicefurther comprises a matrix disposed within the semipermeable membrane.6. The device of claim 1, wherein the device further comprises a tetheranchor.
 7. The device of claim 1, wherein said growth factor is selectedfrom the group consisting of brain derived neurotrophic factor (BDNF),neurotrophin-4 (NT-4), ciliary neurotrophic factor (CNTF), Axokine,bFGF, IGF I, IGF II, TGFβ II, Midkine, IL-1β, nerve growth factor (NGF),IL-2/3, IL-6, neurturin (NTN), Neublastin, VEGF, glial cell derivedneurotrophic factor (GDNF), platelet derived growth factor (PDGF), lensepithelium derived growth factor (LEDGF), and pigment epithelium derivedgrowth factor (PEDF).
 8. The device of claim 7, wherein said growthfactor is ciliary neurotrophic factor (CNTF).
 9. The device of claim 7,wherein said growth factor is glial cell derived neurotrophic factor(GDNF).
 10. A method for inhibiting retinal degradation, comprisingimplanting into the eye of a recipient host with retinal degeneration animplantable cell culture device, the device comprising: (a) asemipermeable membrane permitting the diffusion of a growth factortherethrough; and (b) ARPE-19 cells that are genetically engineered tosecrete the growth factor; wherein the device releases a therapeuticallyeffective amount of said growth factor into the eye of the hostrecipient, thereby inhibiting the retinal degradation.
 11. The method ofclaim 10, wherein the growth factor is ciliary neurotrophic factor(CNTF).
 12. The method of claim 11, wherein the therapeuticallyeffective amount of CNTF is between 100-350 ng/million cells/24 hr(ng/M/d).
 13. The method of claim 11, wherein the therapeuticallyeffective amount of CNTF is about 1.62±0.4 ng/device/24 h.
 14. Themethod of claim 10, wherein the retinal degradation is caused by adisorder selected from the group consisting of Retinophathy ofPrematurity, Diabetic Retinophathy, Age-related Macular Degeneration,Glaucoma, Retinitis Pigmentosa, Cataract Formation, Retinoblastoma, andRetinal lschemia.